Device for raising or cultivating cells in a container-like receptacle

ABSTRACT

A device for raising or cultivating cells in a container-like receptacle, comprises a base and at least one lid. The at least one lid is connected to the receptacle in a pressure-tight manner. The receptacle or the lid is provided with at least one connector drilling for the introduction and/or extraction of culture medium and/or oxygen.

This application is a national stage completion of PCT/EP2003/000211filed Jan. 11, 2003 which claims priority from German application SerialNo. 102 01 259.8 filed Jan. 15, 2002.

FIELD OF THE INVENTION

The invention relates to a device for raising or cultivating cells in acontainer-like receptacle with comprises a base and at least one lid.

BACKGROUND OF THE INVENTION

For laboratory requirements, it is known to raise or cultivate cellcultures in a shallow receptacle or dish, the cells simply being placedin the receptacle and culture medium being added. A lid is then placedon the receptacle.

A disadvantage is that this method can only be used for smallquantities. In particular, the known system is not suitable forcultivating or raising cells in batches. Moreover, it is impossible toachieve in vivo conditions, and sterility is not guaranteed.

An alternative to this was to use a closed system which comprised areceptacle with a lid or closure piece and which permitted sterility.However, a disadvantage of this was that removing the cell culture wasvery laborious and time-consuming.

It is therefore an object of the present invention to improve a deviceof the type mentioned at the outset in such a way that it can be used ina highly versatile manner for raising or cultivating cells, particularlyin large-scale operations, the aim being to achieve as far as possiblein vivo conditions and sterility. Moreover, after they have beencultivated, the cell cultures should be able to be removed from thereceptacle without great effort and without being damaged.

SUMMARY OF THE INVENTION

According to the invention, this object is achieved by the features inthe characterizing part of claim 1.

In the device according to the invention, the cells in the receptacleare no longer “left to themselves”, and instead an active process takesplace in practice. On the one hand, culture medium can be introducedcontinuously or intermittently, and, on the other hand, it is possiblefor the cell cultures forming to be acted upon with pressure. Thispressure actuation can even be effected with alternating pressure inorder to simulate natural conditions as far as possible.

The cells can be raised or cultivated as separate cell cultures. In thesame way they can also be formed on structures in order to produceimplants.

In the receptacle according to the invention, the cells can in this wayalso be exposed to shearing forces or pressure forces. With the deviceaccording to the invention, a wide variety of cell cultures can beraised or cultivated in a highly versatile manner.

The receptacle itself can also be provided for this purpose variouslywith one or two lids. Likewise, a common connector bore can be providedfor the introduction of culture medium and for its return. Of course,separate connector bores are also possible, in which case parallel flowsor through-flows are possible depending on the arrangement of theconnector bores.

By virtue of the configuration according to the invention with one ortwo lids or a lid and base, and between them a receptacle which cansimply be a cylinder open at the top and bottom, the cell cultures caneasily be removed from the receptacle, after they have been treated orraised, without great effort and without damaging them.

Since according to the invention the cells are raised or cultivated onthe lower lid or base or also under the upper lid, e.g. on a frameconnected to the upper lid, the cell cultures can be easily removed fromthe receptacle once they are ready. In any event, good accessibility isafforded by the removable lids or base.

For pressurization, the receptacle, e.g. the circumferential wall of thecylindrical receptacle, can also be made elastic.

The connector bore or connector bores can be arranged in one lid or, iftwo lids are present, in both lids. Likewise, it is also possible toform the connector bores in the cylindrical middle part. The number andarrangement of the connector bores depends on the application and on thecells which are to be raised or cultivated.

For a tight connection between the lid or lids and the receptacle, clampconnections, sealing rings or threaded connections with internal andexternal threads can be provided.

Very good sealing conditions and thus pressure conditions are obtainedif the lid or lids are provided with extension rings which thensealingly enclose the cylindrical middle part of the receptacle from theoutside.

If the device according to the invention is to be subjected to a rollingor turning movement, tensioning rings can be fitted laterally onto thedevice, which tensioning rings grip the lid or lids and the receptacle,and a turning or rolling means can then be applied to them.

A wide variety of pressurizing means can be used to subject the interiorof the receptacle forming the cell culture chamber to pressure. Suitablefor this purpose are, for example, cylinder/piston units which can alsooperate in pulsed mode for alternating pressure loads.

If necessary, the receptacle can also be designed as a two-chambersystem so that two different cells or two identical cells can becultivated or raised separate from one another.

In this case, it is advantageous if the lid of the receptacle isprovided with a suspension means on which a platform for receiving cellsis arranged. In this way, one cell type is raised on the platform, whileanother cell type can be cultivated on the base of the receptacle.

If necessary, the receptacle or the cylindrical circumferential wall ofthe receptacle can be made porous or gas-permeable, so that in this waytoo culture medium and/or a gaseous medium, for example air or oxygen,can be delivered from this side.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of example, with referenceto the accompanying drawing in which:

FIG. 1 shows a cross section through a first embodiment of a device witha receptacle and a lid;

FIG. 2 shows a cross section through a device with a cylindrical middlepart and an upper lid and lower lid;

FIG. 3 shows a similar configuration to the one in FIG. 2, with an inletconnector and an outlet connector being arranged in the upper lid;

FIG. 4 shows a further embodiment of the device according to theinvention;

FIG. 5 shows a device according to the invention with an upper lid and alower lid, each with external thread, and a cylindrical middle part withinternal thread;

FIG. 6 shows an embodiment with extension rings on an upper lid and alower lid;

FIG. 7 shows a similar configuration to the one in FIG. 6, with a clampconnection;

FIG. 8 shows a similar configuration to the one in FIGS. 6 and 7, withthreaded connections;

FIG. 9 shows an embodiment with two lateral tensioning rings;

FIG. 10 shows an embodiment similar to the embodiment in FIG. 9, with apressurizing means;

FIG. 11 shows an embodiment with a suspension means in the upper lid,with a cylindrical middle part and a lower lid;

FIG. 12 shows a similar configuration to the one in FIG. 11, in somewhatsimpler form with just an upper lid;

FIG. 13 shows a configuration with a pressure force generated bymagnetic forces;

FIGS. 13 a, 13 b and 13 c show different pressure disk profiles;

FIG. 13 d shows a mineral matrix for bone replacement as supportstructure with two pressure disks;

FIG. 14 shows a configuration with expandable elements for generatingpressure forces;

FIG. 15 shows a configuration with hydraulic or pneumatic elements forgenerating pressure forces;

FIG. 16 shows a similar configuration to the one in FIG. 15;

FIG. 17 shows a further configuration similar to those in FIGS. 15 and16;

FIG. 18 shows a configuration with a gas-permeable membrane as base;

FIG. 19 shows a configuration with a three-chamber system;

FIG. 20 shows a similar configuration to the one in FIG. 19.

DETAILED DESCRIPTION OF THE INVENTION

According to the illustrative embodiment in FIG. 1, which shows thebasic configuration of the device according to the invention, areceptacle 1 is provided which has an external thread 2. A lid 3 with aninternal thread 4 is screwed onto the receptacle 1, and a sealing ring 5forms a pressure-tight closure of a cell culture chamber 6 for cells 7provided in the interior of the receptacle 1.

The lid 3 is provided with an inlet connector bore 8 and an outletconnector bore 9 in order to introduce culture medium and if appropriatealso oxygen into the cell culture chamber 6 via corresponding lines ortubes.

For easier handling, a tray 10 can be inserted into the receptacle 1 soas to make it easier to insert and remove the cells 7 which are to becultivated or raised. For this purpose, beads, flanges 11 or the likewhich are provided in the upper area of the tray 10 can also serve foreasier removal and easier insertion. The tray 10 shown only in FIG. 1can of course also be provided in the same way or in a similar way inthe other illustrative embodiments.

FIG. 2 shows a configuration where the receptacle 1 forms a cylindricalmiddle part which can be closed with the upper lid 3 in the same way asin the illustrative embodiment according to FIG. 1. Provided on theunderside there is a further, lower lid 12 which forms the base of thereceptacle and which likewise has an internal thread 4 which interactswith the external thread 2 of the middle part, said middle part in thiscase being provided with two external threads 2, unless one continuousthread is present. As will also be apparent from FIG. 2, inlet andoutlet connector bores 8 and 9 are provided in both lids 3 and 12.

FIG. 3 shows a similar configuration to the one in FIG. 2, with an inletconnector bore 8 and an outlet connector bore 9 being arranged only inthe upper lid 3. In the same way as in the illustrative embodimentaccording to FIG. 2, sealing rings 5 are provided on both ends of thecylindrical middle part of the receptacle 1.

FIG. 4 shows an embodiment in which the cylindrical middle part asreceptacle 1 is provided with an internal thread 2′ which cooperateswith an external thread 4′ of the upper lid 3 and of the lower lid 12.Here too, sealing rings 5 are provided on both ends of the receptacle 1.In this case, an inlet connector bore 8 is provided in the upper lid 3,and an outlet connector bore 9 is provided in the lower lid 12.

FIG. 5 shows a similar configuration to FIG. 4. The main difference issimply that the inlet connector bore 8 and the outlet connector bore 9are arranged opposite one another in the cylindrical middle part of thereceptacle 1.

FIG. 6 shows an embodiment with a cylindrical middle part as receptacle1, an upper lid 3, and a lower lid 12. Both lids 3 and 12 each have anexternal thread 4′ which cooperates with internal threads 2′ of thereceptacle 1. In addition, both lids 3 and 12 are provided with radialextensions 13 from whose outer ends extension rings 14 extend axiallyinto the receptacle 1, parallel to the longitudinal axis of thereceptacle 1. The extension rings 14 enclose the outer wall of thereceptacle 1 and, together with additional sealing rings 5′, constitutea pressure-tight closure for the cell culture chamber 6. The upper lid 3is provided with a common connector bore 8, 9 for the introduction andwithdrawal of culture medium.

FIG. 7 shows a similar embodiment to the one in FIG. 6. The maindifference is simply that, instead of a threaded connection 2′, 4′, apressure-tight closure of the cell culture chamber 6 is created by clampconnections between the extension rings 14 and the outside wall of thereceptacle 1.

FIG. 8 shows an embodiment similar to those in FIGS. 6 and 7. The maindifference here is that the threaded connection between the receptacle 1and the lids 3 and 12 is formed by internal threads 4′ in the extensionrings 14, these cooperating with external threads 2′ in the receptacle1.

FIG. 9 shows an embodiment with a receptacle 1 and an upper lid 3,similar to the embodiment according to FIG. 8, but instead of a commonconnector bore 8, 9 for the introduction and withdrawal of culturemedium, an inlet connector bore 8 and an outlet connector bore 9 arearranged in the circumferential wall of the receptacle 1. In addition,FIG. 9 shows two lateral tensioning rings 15 which are pushed round thecontainer-like receptacle 1 and the lid 3 in the arrow direction so thatthe unit consisting of receptacle 1 and lid 3 can be turned or rolled inarrow direction 16 about the transverse axis with the aid of a turningor rolling means (not shown).

FIG. 10 shows a similar configuration to the one in FIG. 9. In thiscase, a separate cell culture chamber 6 is formed in the interior of thereceptacle 1. Instead of a cell culture chamber 6, it is also possibleto provide a structure on which the cells 7 are raised. The separatecell culture chamber 6 or the structure is in this case pressurized viaa pressurizing means 17 in the form of a cylinder/piston unit.

The inlet connector bore 8, which can be shut off at the entry point bya check valve 19, opens into a piston chamber 18 of the cylinder/pistonunit 17. The culture medium introduced through the inlet connector bore8 is pressurized by a piston 20 of the cylinder/piston unit 17, and thispressure continues into the interior of the receptacle 1. Culture mediumis withdrawn via an outlet connector bore 9 on the side of thereceptacle 1 directed away from the inlet. When the interior of thereceptacle 1 is to be subjected to an overpressure, which may ifappropriate be alternating, the return flow of culture medium will inthis case be constricted or the outlet connector bore 9 correspondinglyshut off.

Instead of supplying culture medium via the inlet connector bore 8, aseparate bore can also be provided for this in one of the two lids 3 or12 or in the circumferential wall of the receptacle 1. In this case, itis also possible to use gas, e.g. air, to pressurize the interior of thereceptacle 1 and thus the cell culture chamber 6.

FIG. 11 shows an embodiment with an upper lid 3 and a lower lid 12 and acylindrical middle part of a receptacle 1. In this case, the upper lid 3is provided with a suspension means in the form of several rods 21 whichare distributed about the circumference and which extend, parallel tothe longitudinal axis of the receptacle 1, into the interior of thereceptacle 1. Secured at the lower end of the rods 21 there is aplatform 22 on which the cells 7 to be cultivated or raised arearranged. The inlet connector bore 8 and the outlet connector bore 9 caneach be arranged in the circumferential wall of the receptacle 1. Ofcourse, it is also possible to arrange them in one of the two lids 3 or12, as is indicated by broken lines. In this case too, separateconnector bores are of course also possible for inlet and outlet.

The advantage of the embodiment with the suspension means formed by therods 21 is that in this way the cells 7 are easier to insert into thereceptacle 1 and remove therefrom.

If necessary, the connection of the rods 21 to the platform 22 can bemade detachable. Detachability can be achieved, for example, by a clipconnection, which also provides for easier handling of the device.

FIG. 12 shows an embodiment which is similar to the embodiment shown inFIG. 11. As will be noted, it only has an upper lid 3 and a receptacle 1with base 23, as is also present in the other embodiments with just onelid 3. The platform 22 in this case is again connected to the upper lid3 via rods 21.

A further advantage of the embodiments according to FIGS. 11 and 12 isthat the receptacle base 23 or inner side of the lid 12 provides anadditional possibility for raising or cultivating cells 7. In this way,a two-chamber system for cultivation of two cell cultures is created.

Instead of inlet connector bores 8 and outlet connector bores 9 forculture medium, culture medium can of course also be introducedcontinuously into the receptacle 1, and the inlet connector bores 8 andoutlet connector bores 9 then serve only for oxygen supply.

Alternatively, it is also possible to provide separate connector boresfor oxygen and culture medium.

The platform 22 can be designed as a solid unit. Alternatively, it isalso possible for this purpose to provide a membrane, e.g. a porousmembrane, which allows oxygen to pass through.

FIGS. 13 through 20 show further configurations of the invention, thebasic structure of the device with receptacle 1 and both lids 3, 12and/or base 23 having been retained, for which reason, to keep matterssimple, only the relevant reference numbers have been repeated in thefollowing description of these figures.

FIG. 13 shows a receptacle 1 in which a magnetic means 24, e.g. a magnetcoil through which current flows, is incorporated in the area of theupper lid 3. Under the magnetic means 24 there is a magnetizablepressure disk 25 which is connected elastically to the receptacle 1 in amanner not shown in detail.

Through a movement of the pressure disk 25 caused by activation of themagnetic means 24, generated by alternate current directions forexample, an internal pressure load is exerted on the cells 7.

FIG. 13 a shows a plan view of the profile of a pressure disk 25, smallopenings 26 being provided so that culture medium located in the insideof the receptacle 1 can pass through.

FIGS. 13 b and 13 c show alternative pressure disks 25 in the form of amesh structure or grid structure so that culture medium can passthrough.

Of course, the magnet coil acting as magnetic means 24 can also bearranged outside the lid 3, above the latter. In this configuration, thelid 3 of course has to be made of nonmagnetizable material, e.g.plastic. In this case, suitably large magnetic means 24 can be providedand correspondingly high pressure forces generated.

FIG. 13 d shows a configuration of an implant, cartilage profiles 27being arranged on a mineral matrix for bone replacement as supportstructure 27 a. Here, two support structures are provided which arearranged one above the other and on each of which a pressure disk 25 isarranged. The mineral matrix can, for example, be a bone structure, e.g.of calcium phosphate.

The mineral matrix can also have other profiles as are required forimplants, e.g. joint structures. It is also possible, of course, todeviate from the circular shape. The same also applies in principle tothe receptacle 1.

FIG. 14 shows a configuration with expandable elements 28 which axiallydisplace a plate 29 arranged movably in the receptacle 1 or the lid 3,in the same way as the pressure disks 25, and thus can exert alternatingpressure forces on the cells 7. The expandable elements used can, forexample, be shape-memory metals or plastics which deform and then returnto the original shape. Thus, for example, there are also plastics whichcan expand through electrical change. Elements with shape-memoryfunction react, for example, to certain temperatures or to ultrasoundand in this way alter their state, thus generating a movement of theplate 30. Spring devices are also possible, as are motors withaccumulators or batteries.

FIG. 15 likewise shows internal pressurization of cells 7 by a hydraulicor pneumatic means 30 arranged in the receptacle 1 or in the lid 3. Aswill be noted, the means 30 has a movable film, plate or membrane 31,behind which a hydraulic liquid or a gas acting as fluid is located. Thehydraulic liquid or the gaseous medium is subjected to alternatingpressure by a pressurizing means P (not shown in detail), as a result ofwhich alternating pressure loads are exerted on the cells 7. Instead ofan elastic plate or membrane 31, a balloon can also be used, ifrequired, in order to obtain the possibility of greater variation. Forexample, all the walls of the receptacle can be covered on the innerside by such a bag or balloon, in which case the implant or cellcultures are located in the inside. In this way, an alternating pressureload is exerted all round from outside.

FIG. 16 shows a similar configuration in which the cells 7 lie in a gel32.

FIG. 17 likewise shows a similar configuration to the one in FIG. 16,the sealing between the upper lid 3 and the lower lid 12 being obtainedby sealing rings 33 and 34.

FIG. 18 shows a configuration in which the base of the receptacle isformed by a gas-permeable membrane 35, e.g. of PTFE or silicone. Theimportant thing is that oxygen can pass into the receptacle 1 throughthe membrane 35. In this configuration too, an implant growing in theinterior of the receptacle can be easily removed. Such a configurationis suitable, for example, for growing epidermis 36, i.e. the top layerof the skin, on the membrane 35. The cells are supplied with air throughthe gas-permeable membrane 35. The aim here is to have the cells growfrom the underside upward. In addition, a dermis 37 is then applied ontop, the cells being arranged or cast in a collagen structure or infibrin. At the top, above the dermis 37, there is a clean area 38 inwhich air, oxygen and/or carbon dioxide is introduced. Pressurization isalso possible here.

In this way, a skin structure is obtained as exists in reality. If sorequired, various other cells can also be added, for example endothelialcells in order to achieve vascularization on the top face.

To achieve sterility and protection for the membrane 35, a coveringstructure 39 can, if required, be fitted tightly or screwed on at thebottom.

On the inside, the receptacle can be provided with a peel-off film 40for transplantations. On completion of the cell cultivation, thecovering structure 39 is in this case removed and the film 40 is thenpeeled off like a plaster. In this way, the implant can be easilyremoved and then used. The dermis 37 then lies in the correct positionon the wound and, when the film is peeled off, the epidermis 36 lies ontop.

Instead of a covering structure 39, a chamber with lateral openings canalso be provided, in which case a sterile filter is fitted.

FIG. 19 shows a similar configuration to the one in FIG. 18. Here, bycontrast, a multi-chamber system is formed, with an upper chamber 41, amiddle chamber 42 and a lower chamber 43. Separate inlets and outletscan be provided for all three chambers 41, 42, 43. Thus, for example,the epidermis 36 can be arranged in the chamber 41, the dermis 38 can bearranged in the chamber 42, and the lower chamber 43 is used for thesupply of air or oxygen. Culture medium can be introduced into thechamber 41 in a first step. Then, in a second phase after the cells 7have grown, if differentiation is desired, oxygen or air can beintroduced into the chamber 41 so that the epidermis cells 36 acquire adry environment and are then able to dry out and keratinize. In thisway, the upper layer of skin is then formed. If one then wishes toimplant the whole arrangement, the receptacle can be easily opened andthe implant removed.

If the cells 7 arranged in the chamber 42 require a support structure, aporous support or membrane 44 can be provided on the underside andconstitutes the division between the chamber 42 and the chamber 43.

1. A device for raising or cultivating cells in a container-likereceptacle (1), the device comprising: a cylindrical middle part of thedevice being closed off at a top by an upper lid (3) and a lower lid(12) which forms a base of the receptacle (1), wherein the upper lid (3)and the lower lid (12) are connected to the middle part in apressure-tight manner and the upper lid (3) and the lower lid (12) areeach provided with at least one inlet bore (8) for one of anintroduction and a withdrawal of a culture medium and oxygen, the upperand the lower lids (3, 12) and the middle part are connected to oneanother by mating internal and external threaded connections (2, 4), andeach threaded connection is provided with at least one sealing ring, andthe upper lid (3) and the lower lid (12) each include an extension ring(14) having an extension ring region extending beyond the threadedconnections (2, 4) to at least partially enclose the cylindrical middlepart and each extension ring region including at least one sealing ringlocated between the middle part and the extension ring region toadditionally seal the middle part.
 2. A device for raising orcultivating cells in a container-like receptacle which comprises: a basereceptacle (1); and at least an upper lid (3); wherein the upper lid (3)is connected to the base receptacle (1) in a pressure-tight manner bymating internal and external threaded connections of the upper lid (3)and base receptacle (1), and each threaded connection is provided withat least one sealing ring, and at least one of the base receptacle (1)and the upper lid (3) is provided with at least one inlet bore (8) forone of introduction and withdrawal of a culture medium and oxygen; atleast one resilient lateral tensioning ring (15) encircles both anexterior surface of the base receptacle (1) and an exterior surface ofthe upper lid (3) to retain the upper lid (3) in sealing engagement withthe base receptacle (1) when the container-like receptacle is rotatedabout a transverse axis; and a pressurizing device coupled to the devicefor raising or cultivating cells for exerting pressure on the cells (7)being cultivated within the device.
 3. A device for raising orcultivating cells in a container-like receptacle (1) which comprises abase; and at least one lid, wherein the at least one lid (3) isconnected to the receptacle (1) in a pressure-tight manner, and thereceptacle (1) or the at least one lid (3) is provided with at least oneinlet bore (8) for one of the introduction and withdrawal of culturemedium and oxygen, and a magnetizable pressure disk (25) is arranged inthe receptacle (1) and moved by a magnetizing means (24) in order toexert pressure internally on the cells (7).
 4. The device as claimed inclaim 3, wherein the pressure disk (25) is provided with holes (26). 5.The device as claimed in claim 3, wherein the pressure disk (25) has agrid or mesh structure.
 6. The device as claimed in claim 3, wherein thecells (7) are arranged on a support structure (27 a) which is acted uponby the pressure disk (25) from one or both sides.
 7. A device forraising or cultivating cells in a container-like receptacle (1) whichcomprises a base; and at least one lid; wherein the at least one lid (3)is connected to the receptacle (1) in a pressure-tight manner, and thereceptacle (1) or the at least one lid (3) is provided with at least oneinlet bore (8) for one of the introduction and withdrawal of a culturemedium and oxygen, a pressurizing means is located within a structure ofthe receptacle (1) for exerting fluid pressure on both the culturemedium and the cells (7) being cultivated within the device so that apressure load is exerted on the cells all round from outside.
 8. Thedevice as claimed in claim 7, wherein the pressurizing means is formedof expandable elements (28).
 9. The device as claimed in claim 7,wherein the pressurizing means (17) is designed as a cylinder/pistonunit.
 10. The device as claimed in claim 7, wherein the pressurizingmeans (17) subjects an interior of the base receptacle (1) containingthe cells (7) to alternating pressure loads.
 11. The device as claimedin claim 7, wherein the pressurizing means includes a movable film,plate or membrane (31) arranged in the receptacle (1).
 12. The device asclaimed in claim 7, wherein the pressurizing means, which exerts thepressure load on the cells, is separate and independent of the culturemedium.
 13. The device as claimed in claim 7, wherein the pressurizingmeans exerts an alternating pressure load on the cells.
 14. The deviceas claimed in claim 7, wherein the pressurizing means includes a movablefilm, plate or membrane (31) arranged in the receptacle (1) and one of ahydraulic liquid and a gaseous medium is located on one side of the oneof the movable film, plate or membrane (31) while the culture medium andthe cells (7) are located on an opposite side of the one of the movablefilm, plate or membrane (31).
 15. The device as claimed in claim 7,wherein the pressurizing means is one of a hydraulic liquid and agaseous medium.
 16. The device as claimed in claim 7, wherein thepressurizing means comprises one of a bag and a balloon and the culturemedium and the cells (7) are located within one of the bag and theballoon.
 17. The device as claimed in claim 14, wherein the culturemedium is a gel.